The long-term goal of this research proposal is to understand the role of pre- and/or post-transcriptional regulation of glycosaminoglycans' (GAGs) biosynthetic enzymes, their subcellular compartmentation and that of their substrates. Towards achieving these goals we will: (a) conclude our studies on the purification, molecular cloning and expression of rat liver Golgi membrane transporters for UDP-N-acetylgalactosamine, UDP-glucuronic acid, adenosine 3' phosphate 5'-phosphosufate and ATP. These proteins appear to be important in regulating the supply of nucleotide substrates in the Golgi lumen and thereby GAG biosynthesis. (b) In collaboration with Dr. R. Horwitz's group (MIT), we will determine which GAG are synthesized in C. elegans and the substrate specificity of putative nucleotide sugar transporters that seem to be involved in its vulva development. (c) Determine the recognition domains for nucleotides, sugars and sulfate of the MDCK transporters for UDP- N-acetylglucosamine and PAPS by using a combination of photolabelling, cross-linking and mutagenesis. Possible complexes in the Golgi membrane between these transporters and the corresponding glycosyl- and/or sulfo-transferases will be explored. (d) Continue to study the role of the above transporters in regulation of GAG biosynthesis in the lumen of the Golgi apparatus by determining the effect of inducible transporter expression and antisense thiooligonucleotides to these transporters. (e) Continue with our studies on the heparin/heparan sulfate N-deacetylases/N-sulfotransferases, key dual catalytic active site enzymes, in the biosynthesis of these important macromolecules. We plan to demonstrate that the N- deacetylase activity is as predicted, towards the amino half of the protein and determine the consequences of the differential expression of each of the above activities on the biosynthesis of heparin versus heparan sulfate. (f) Identify and express homologs to the Golgi ATP transporter as initial steps towards the identification and reconstitution of the endoplasmic reticulum ATP transporter.